Seasonal variations in the heterologous binding of viscacha spermatozoa . A scanning electron microscope study

Seasonal changes in the reproductive activity of the adult male viscacha (Lagostomus maximus maximus) were investigated during the annual reproductive cycle. Assays of heterologous in vitro binding between compatible gametes were used to evaluate the ability of viscacha spermatozoa to achieve primary binding during its annual reproductive cycle. Sperm were collected by mincing cauda epididymis in HECM-3 medium and the sperm concentration and motility were evaluated. Cumulus-free and zona-free oocytes obtained from superovulated hamsters were inseminated in vitro with capacitated sperm suspensions, incubated at 37oC, 5% CO2 for 3 h, and then processed for studies by scanning electronic microscopy. Statistical analysis was used to compare the quantitative differences. The number of spermatozoa significantly decreases during the regression period, while sperm motility was progressive speed in both periods. During the active period elevated sperm binding to cumulus-free and zona-free oocytes was observed, while the binding during the regression period decreased drastically. In both periods, oocyte microvilli covered sperm heads and tails. These results suggest that the ability of viscacha spermatozoa to participate in gamete recognition is profoundly affected. This would likely be related to different functional stages of the spermatozoa and their epididymal microenvironment during the annual reproductive cycle of viscacha. BIOCELL 2005, 29(3): 243-251 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
Fusion between gametes is a key event in the fertilization process, involving the interactions of specific domains of the sperm and egg plasma membranes.Although considerable information has been obtained concerning the structural aspects of mammalian sperm-egg fusion, only recently progress has been made towards the identification of specific molecular components that mediate this event (Yanagimachi, 1988;Myles, 1993).
One of the major sources of the specificity of fertilization in mammals is the zona pellucida, a glycoprotein coat surrounding the egg proper, where speciesspecific gamete recognition and signaling occurs.When the zona is removed, the vitelline surface of the eggs of most mammalian species (e.g., hamster and rabbits) permits the penetration of sperm form heterologous species (Hanada and Chang, 1976).
Mammalian spermatozoa produced in the testis must mature in the epididymis to acquire their fertilizing capability.During fertilization, only the capacitated spermatozoa are capable of recognizing and binding to the zona pellucida or zona free-eggs and undergoing the acrosome reaction (Cross and Meizel, 1989).
In regions with important differences in environmental factors, mammalian reproduction is entrained principally by photoperiod and temperature, and natural selection has produced different adaptations in order to subsist in adversity (Darwin, 1858).Species such as the Dzungarian or Mesocricetys auratus hamsters need to plan the birth of its progeny during an ideal period of temperature and diet.Thus, the reproductive tract is one of the systems most sensitive to these environmental adaptations (Badura et al., 1992;Ferkin and Gorman, 1992;Begay et al., 1993;O' Brien et al., 1993;Gutierrez et al., 1995).
Lagostomus maximus maximus (viscacha) is a seasonal rodent (Llanos and Crespo, 1952).Under natural conditions the adult male shows testicular involution during the shorts days of winter (July-August) and maximum gonadal activity during the long days of summer and autumn (December-March).In the regressed testes, the seminiferous tubules are reduced into cords (Fuentes et al., 1991).These changes are accompanied by a decrease in serum levels of testosterone and testicular concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (Prl) receptors, and a reduced capacity of Leydig cells to synthesize and secrete androgen (Fuentes et al., 1993;Muñoz et al., 1997).The viscacha epididymis accompanies the testes in their profound morphological and biochemical changes, and rhythmic fluctuations of testosterone (Aguilera Merlo et al., 2000).
Biochemical, neural, and endocrine factors and behavioral rhythms play a central role in many different aspects of the reproductive process in animals.These rhythms provide the basis for the temporal organization of reproductive function in individual animals (Turek and Van Cauter, 1994).
In the study reported here, we have shown results obtained through heterologous primary binding between hamster oocytes and viscacha spermatozoa during the seasonal reproductive cycle.

Animals
Twelve adult viscachas (Lagostomus maximus maximus) weighing 4-8 kg were captured in their habitat near San Luis, Argentina (33º 20' south latitude, 760 m altitude) during the periods of maximum gonadal activity (summer-autumn) and gonadal regression (winter).Viscachas were anaesthetized with Nembutal (pentobar-bital) and quickly decapitated.The epididymides were exposed through abdominal incision and a ligature was placed at the limit between corpus and cauda.The ligated cauda was removed and immersed in HECM-3 medium (Ogura and Yanagimachi, 1993).

Semen processing
Sperm collected from viscacha were handled essentially as described previously (Bleil and Wassarman, 1983).Sperm were collected by mincing cauda epididymis in 2.0 ml of HECM-3 medium and epididymal tissue was removed by centrifugation.Aliquots of 0.5 ml to 1.0 ml were put in plastic culture tubes and 2 volumes of HECM-3 medium were added.The sperm suspension was incubated at 37ºC in air supplemented with 5% CO 2 during 1 h.Then, the supernatant containing motile spermatozoa (swim-up fraction) was transferred to new culture tubes and incubated at 37ºC in air supplemented with 5% CO 2 for 4-5 hr.The swim-up fraction was centrifuged 3 times at 500 g for 5 min in conical tubes (Falcon 2063).The resulting pellets were resuspended in fresh medium to obtain a final concentration of 1x10 6 sperm/ml and 0.5x10 6 sperm/ml (Who Protocol, 1999).

Oocyte preparation
Oocytes were collected from the oviducts of superovulated golden hamsters between 15 and 17 h after injection of HCG.The oocytes were freed from cumulus cells by treatment for 5-10 min in HECM-3, containing 0.1% bovine testicular hyaluronidase (Sigma Chem.Co, St Louis MO).After being rinsed four times in fresh medium, zona-free oocytes were obtained for treatment with 1% bovine pancreas trypsin type III (Sigma Chem.Co, St Louis MO) for a few minutes.The oocytes were rinsed thoroughly in fresh medium five times, transferred into 100 μl HECM-3 medium and placed under mineral oil in a plastic Petri dish at 37°C to be used within 30 min.Abnormal oocytes were discarded (WHO Protocol, 1999).

Sperm-oocyte interaction
Drops of 100 μl of the capacitated viscacha sperm suspension (from both regressive and high activity periods of the reproductive cycle), diluted to a concentration of 0.5x10 6 sperm/ml and 1x10 6 sperm/ml in HECM-3 medium were placed under mineral oil in Falcon 3001dishes (35x10mm).Approximately, fifteen cumu- lus-free and ten zona-free oocytes were introduced into each sperm suspension drop and incubated for 3 h at 37ºC in air supplemented with 5% CO 2. (WHO Protocol, 1999).The sperm-oocytes complexes were washed 3 times in fresh medium to eliminate weakly adhered spermatozoa.

Scanning procedure
Bound sperm-oocytes were fixed with 1% glutaraldehyde in PBS buffer (pH 7.4) for 2 h at 4°C, and then were washed repeatedly to eliminate excess glutaraldehyde.The bound gametes were placed on metal grills (covered with 1% gelatin), processed by dehydration through graded alcohol-acetone concentrations, critical point drying and gold evaporation in a sputter device for scanning electron microscopy (SEM), and observed in an Autoscan Siemens ETEC.

Statistical Analysis
All the data were expressed as means ± SEM.Differences between groups were evaluated using Kruskal-Wallis test (nonparametric ANOVA) followed by pairwise comparisons using a Dunn test.A p-value < 0.05 was accepted as statistically significant.

Sperm Analysis
Spermatozoa from cauda epididymides were able to develop progressive motility.These patterns of movement were observed both in active and regressive gonadal periods.The sperm quantification was 470x10 6 (±12.5x 10 6 ) during the active period and 70.8x10 6 (±6.27x 10 6 ) in the gonadal regressive period.

Binding of viscacha sperm to cumulus-free hamster oocytes Active Period
During the period of maximum gonadal activity (summer-autumn), viscacha sperm bound avidly to zona pellucida (ZP) oocytes forming the sperm-oocyte complexes that rotate in the medium after initiating the incubation period (Fig. 1a).The number of viscacha sperm binding to each ZP of hamster oocyte was elevated at a concentration of 1x10 6 sperm/ml, while a significant decrease of viscacha sperm binding to ZP was observed at concentration of 0.5x10 6 sperm/ml (Table 1).Most sperm remained tightly bound after washing the vis-  cacha sperm-oocyte complex.There was no penetration of the ZP by sperm during the incubation time.All the ZP expressed affinity for gametes of the heterologous specie.Nonetheless, some areas of the ZP remained free of sperm (Fig. 3).

Regressive Period
Viscacha sperm obtained during the period of gonadal regression (winter) showed elevated motility, but little recognition by male gametes to ZP of hamster oocytes was observed in this period compared to the active period (Table 1).Areas completely free of sperm were observed (Fig. 1b).The flagellar activity of the sperm produced displacement and rotation of the oocytes as in the active period.There were no signs of fertilization at time of incubation.

Binding of viscacha sperm to zona-free hamster oocytes Active Period
During the active period of the seasonal viscacha reproductive cycle, sperm showed a high binding capacity for zona-free hamster oocytes.The intense flagellar activity of male gametes displaced and rotated zonafree oocytes in the medium during many minutes after initiating incubation.An elevated number of male gametes were bound to plasma membrane oocytes at concentrations of 1x10 6 sperm/ml, while a significant decrease of male gamete binding to zona-free oocyte was observed at 0.5x10 6 sperm/ml (Table 1).The number of viscacha sperm binding to each zona-free oocyte of unfertilized hamster was so high that it was difficult to count all of the bound sperm in one plane of focus (Fig.

FIGURE 3.
Detail at greater magnification of the interaction between viscacha spermatozoa and hamster oocyte zona pellucida.X 7,000 2a).There were no changes in the amount of sperm binding to zona-free oocytes after washing the viscacha sperm-oocyte complex.The oocyte microvilli showed a strong relation with the heads and sperm tails (Fig. 4).

Regressive Period
During the gonadal regression period of viscacha, a reduced binding of sperm to zona-free oocytes was observed (Fig. 2b).Different concentrations of sperm showed significant differences in the number of bound spermatozoa to zona-free oocytes.On the other hand, the oocyte microvilli showed strong adhesion to the heads and tails of bound spermatozoa, regardless of the repeated washed in fresh medium to eliminate weakly adhered spermatozoa.
In both periods of seasonal reproductive cycle no signs of fertilization were observed.

Discussion
The data obtained in the present work clearly shows a high gamete interaction between viscacha sperm and hamster oocyte with and without zona pellucida during the period of maximum gonadal activity, while this capacity of primary binding between gametes diminishes significantly in the period of testicular and epididymal regression.
During the period of gonadal regression, the population of viscacha spermatozoa demonstrated a reduced binding to zona pellucida and denuded oocytes, although they did not show any significant difference in the parameters of morphology, motility and vitality between the periods studied (Aguilera Merlo et al., 2000).Generally, this is a point of substantial controversy.Some authors propose a slight relation among these standardized parameters and the ability of the sperm to penetrate the ZP in vitro (Martinez et al., 1996;Hammitt et al., 1989).Moreover, it has been proposed that the lack of correlation among the conventional sperm parameters and assays of gamete binding suggest that these assays measure different aspects of the viability and fertilizing capacity of the spermatozoa (Jeyendran et al., 1984).
Gamete recognition or primary binding between cells of the same species needs glycoproteins associated with the oocyte zona pellucida that recognize and establish a chemical link with complementary protein receptors of the spermatozoon (Bedford, 1991;Yanagimachi, 1994).
Heterologous inter-gamete binding occurs only rarely.In vitro analysis of species specificity of fertilization indicate that sperm capacitation and the physiological affinity between the sperm of one species and the vitellus of another may be important limiting factors, but the zona pellucida appears to be a major block to interspecific fertilization (Hanada and Chang, 1976;Juneja et al., 1998).
On the other hand, the molecular consolidation and capacitation of spermatozoa is carried out in the epididymis, whose epithelium establishes the particular environment necessary to confer on the spermatozoon their fertilizing ability (Serre and Robaire, 1998).In humans it has been observed that the epididymal spermatozoa of proven fertile men or patients with a normal epididymis acquire the ability to recognize, bind to and fuse with oocytes.Spermatozoa retrieved from the initial segment or caput region failed to bind or penetrate zonafree hamster oocytes while those from the cauda region were successful (Moore et al., 1983).
This finding permits us to suggest that studies of binding amongst gametes is a good measure to evaluate the maturation of epididymal spermatozoa obtained during extreme periods of activity and gonadal (epididymal) regression in seasonally reproducing animals, such as our experimental model the Lagostomus maximus maximus (Fuentes et al., 1991).Furthermore, heterologous in vitro fertilization (IVF) is an attractive method for evaluating the fertilizing capacity of sperm samples in rare or wild species because it does not require the use of valuable homologous gametes (Soler and Garde, 2003).
The variable capacity of heterologous binding observed in the viscacha spermatozoa might indicate that these spermatozoa present different functional stages depending on the epididymal epithelial cells.Observations obtained previously in our laboratory demonstrated that Leydig cells in the testes of viscacha are in the full process of synthesis during the active period, while in the period of gonadal regression the lowest levels of circulating testosterone coincide with the presence of hypotrophic Leydig cells with evident signs of nuclear and cytoplasmic degeneration, and a decreased testicular concentrations of LH, FSH and PRL receptors (Fuentes et al., 1993;Muñoz et al., 1997).In agreement with Orgebin-Crist et al. (1975), we also think that changes in the androgen levels can alter the capacity of the viscacha epididymis to store spermatozoa by affecting ion and protein profiles of the fluid found in the cauda epididymal lumen.
On the other hand, perhaps the epithelium of the epididymis does not totally lose the ability to stimulate the maturity of male gametes, or alternatively, the epididymis of viscacha maintains stored spermatozoa with morphological features and functional fertilizing capacities obtained during a previous active period (Aguilera Merlo et al., 2005).With regards to this latter possibility, it has been estimated that sperm viability is retained in the cauda epididymidis for 2-3 weeks in animals or even longer (Turner, 1995;Moore, 1996).
In the future, more studies will be needed to establish the molecular and biochemical causes of the highly differential binding between viscacha spermatozoa and hamster oocytes throughout the annual reproductive cycle of viscacha.

FIGURE 2 .
FIGURE 2. Image of zona-free hamster oocyte with viscacha spermatozoa.a: Period of maximum gonadal activity.X 2,500.b: Period of gonadal regression.X 2,500

FIGURE 4 .
FIGURE 4. Detail at greater magnification of the surface of the zona-free hamster oocyte with heads of spermatozoa included among the oocyte microvilli.X 11,000

TABLE 1 . Number of spermatozoa strongly bound to hamster oocytes (range, mean ± SEM) during the seasonal reproductive cycle of the viscacha. Cumulus free and zona-free oocytes of hamster were co-incubated with two concentrations of viscacha spermatozoa
Values between columns with significant difference (p< 0.001).